Process for the production of an antibiotic substance 2&#39;-amino-2&#39;-deoxy-kanamycin in higher yield

ABSTRACT

This invention relates to processes for the production of an antibiotic substance 2&#39;&#39;-amino-2&#39;&#39;-deoxy-kanamycin in higher yield. More particularly, this invention relates to the processes for the production of 2&#39;&#39;-amino-2&#39;&#39;-deoxy-kanamycin by fermentation using mutants of Streptomyces kanamyceticus identified as ATCC21259, ATCC21260, ATCC21161 and ATCC21268.

United States Patent [1 13,616,243

[72] Inventors Shohei Kawaji [51] Int. Cl Cl2d 9/00 Tokyo; [50] Field of Search 195/80 Toyoaki Kawasaki, Tokyo; Masao Murase, Kawasaki-shi; Shunzo Fukatsu, Tokyo; [56] References Clted Masahiro Abe, Kawasaki-shi; Yoshihisa UNITED STATES PATENTS g w T f lhlo, i q gf g 2,936,307 5/1960 Johnson et al 195/80 x amoru uzu i, o o ama-s I; asa lro Ueda, Kawasaki-shi; Hamao Umezawa, FOREIGN PATENTS y 8 of Japan 8,695 1961 Japan [2 PP 751,478 Primary ExaminerJoseph M. Golian [22] Filed Aug. 9, 1968 Attorney-Mason, Fenwick & Lawrence [45] Patented Oct. 26, 1971 [73] Assignee Meiji Seika Kaisha, Ltd.

Tokyo, Japan [54] PROCESS FOR THE PRODUCTION OF AN ANTIBIOTIC SUBSTANCE 2'-AMINO-2 '-DEOXY- KANAMYCIN IN HIGHER YIELD 1 Claim, 2 Drawing Figs.

[52] U.S. Cl 195/80 ABSTRACT: This invention relates to processes for the production of an antibiotic substance 2-amino-2'-deoxykanamyein in higher yield. More particularly, this invention relates to the processes for the production of 2-amino-2'- deoxy-kanamycin by fermentation using mutants of Streptomyces kanamyceticus identified as ATCC2 l 259, ATCC21260, ATCCZI I61 and ATCC2I268.

PATENTEunm 26 I9?! 3,616,243

SHEET 10F 2 FIG. I

260 280 320 340 360 mp WAVE LENGTH ABSORBANCE PATENTEDUU 261971 SHEET 2 UF 2 3 mmmznz ss 00 0 0 0m 00 9 00 00$ 00 2 003 00 2 00 9 00 2 00m. oo ow ooww 00mm 00mm 0 0 8 000* PERCENT TRANSMISSION 4v 2. so a 2 o 6' on an 6 OH 1 2 2 H ll in investigating the commercial production of kanamycin A by the fermentation, we have, found that the known natural strains such as strain 0-4-1 of Streptomyces kanamyceticus which have been used up to now for the commercial production of kanamycin A usually can produce and accumulate kanamycin A in a very much larger amount than 2-amino-2'- deoxy-kanamycin in the culture.

As result of our further investigations, we have found that the proportion of 2-amino-2-deoxy-kanamycin produced and accumulated in the culture may be improved remarkably and may be recovered therefrom in higher yield when some new mutants of Streptomyces kanamyceticus are cultured in the known manner using an ordinary culture medium which are conventionally employed for the cultivation. Thus, we have further discovered that some new mutants of Streptom yces kanamyceticus which we have now derived from a typical natural strain 0-4-1 of Strepromyces kanamyceticus can exhibit a higher ability to produce 2'-amino-2'-deoxy-kanamycin than the parent natural strain 0-4-1, when they are cultivated in the known culture medium.

These new mutants having intrinsically the greater capability to produce 2'-amino-2'-deoxy-kanamycin have been obtained by treating the known, natural strain 0-4-1 of Streptomyces kanamyceticus with a mutation agent, namely nitrogen mustard or irradiation of ultraviolet rays and subjecting the surviving mycelia or spores to special screening.

These new mutants which intrinsically have the greater capability to produce 2'-amino-2-deoxy-kanamycin include four types of mutants which have been given the laboratory designations A-4-6, 3A6, 18-8 and 19-2, respectively. These new mutants each have little or no ability to utilize xylose, and they are distinguishable from the parent strain 0-4-1 which has a greater capability to utilize xylose.

Variation or mutation of Streptomyces kanamyceticus is naturally expected since such is a common property of an actinomyces. Therefore, when any known strain of Streptomyces kanamyceticus is treated with a known mutation agent such as nitrogen mustard, it may be expected that in addition to the above-mentioned mutants A-4-6,3 AG, 18-8 and 19-2, there are obtained other mutants which intrinsically exhibit the greater capability to produce 2'-amino-2'-deoxy-kanamycin.

According to the present invention, therefore, we provide a process for the production of 2'-amino-2'-deoxy-kanamycin in higher yield, which comprises cultivating a mutant of Streptomyces kanamyceticus which intrinsically exhibits the greater capability to produce 2-amino-2'-deoxy-kanamycin, under aerobic conditions in a usual culture medium containing the ordinary carbon sources and nitrogen sources, until 2'- amino-2-deoxy-kanamycin accumulates in a significant amount in the culture, and then recovering said antibiotic from the culture.

As embodiments of the process of the present invention, there are also provided a process for the production of 2'- amino-2'-deoxy-kanamycin in a higher yield, which comprises cultivating the mutant A-46,3AG, 18-8 or 19-2 of Streptomyces kanamyceticus under aerobic conditions in a usual culture medium containing carbohydrate and nitrogenous nutrient until the 2'-amlno-2'-deoxy-kanamycin accumulates in a significant amount in the culture, and then recovering said antibiotic from the culture.

in the process of the present invention, the cultivation may generally be carried out in the usual manner as in the ordinary methods of cultivating the known actinomycetes. The culture medium containing known kinds of nutritional sources for actinomycetes are useful for the production of 2'-amino-2'- deoxy-kanamycin. Thus, the culture medium may contain the sources of carbon such as starch, sucrose, maltose, glucose and glycerol etc. and the sources of nitrogen such as soybean meal, soluble vegetable proteins, corn steep liquor, peptone, meat extract, nitrates and ammonium salts. The culture medium may also contain other appropriate inorganic salts such as NaCl, MgSO, and such a material which will promote the growth of Streptamyces kanamyceticus, if necessary.

For the production of 2'-amino-2'-deoxy-kanamycin, the cultivation on solid medium is possible, but for the production in large quantity it is better to carry out the cultivation in a liquid medium under aerobic conditions. For the production of the antibiotic on a large scale it is much more preferable to employ a method of deep aerated submerged cultivation. it is possible to use the shake-cultivation and the aerated agitated cultivation in a liquid medium. The cultivation temperature usually may be between 25 and 35 C. and preferably at a temperature of 28 C. or in the vicinity thereof. It has been found that the amount of 2'-amino-2-deoxy-kanamycin accumulating in the culture becomes maximum at the end of 3 to 7 days after the beginning of the cultivation.

2-Amino-2-deoxy-kanamycin which has accumulated in the culture may be recovered therefrom by any one of the known method of using ion-exchange resins, the method of extracting with organic solvents and the method of using a precipitating agent which are usually employed for the purification of the known water-soluble, basic antibiotics. For the commercial production of 2'-amino-2'-deoxy-kanamycin, it is preferred to recover this by a method of adsorption and elution with an ion exchanger, preferably carboxylic acid type cation-exchange resin, or by a method of extraction with an organic solvent containing an appropriate carrier such as paratoluene sulfonic acid or lauric acid. For instance, 2'- amino-2'-deoxy-kanamycin may be recovered by filtering off the solids such as mycelium cake from the fermented broth containing 2'-amino-2'-deoxy-kanamycin, subjecting the filtrate to an adsorption treatment with Amberlite lRC-SO (a registered Trade Name of an ion-exchange resin as produced by Rohm & Haas Co., U.S.A.), eluting with aqueous ammonia, collecting and concentrating the active fractions, subjecting the concentrated fraction to chromatographic separation with Dowex 1X2 (a registered Trade Name of an ion-exchange resin as produced by Dow Chemical Co., U.S.A.), collecting and concentrating the active fractions. The concentrated fraction is again subjected to a chromatographic separation by adsorbing on Amberlite CG-SO and gradient eluting with wateraqueous ammonia. A single fraction containing an active compound, that is, 2-amino-2'-deoxy-kanamycin may be obtained. This fraction is freeze-dried and the resulting powder is crystallized from water-dimethyl formamide to yield the crystals of 2'-amino-2'-deoxy-kanamycin.

2-Amino-2-deoxy-kanamycin may be obtained in the form of colorless needlelike crystals having a melting point of 183-186 C. basic and soluble in water, aqueous methanol, aqueous ethanol, aqueous acetone but insoluble in absolute methanol, absolute ethanol, ethyl acetate, butyl acetate, ethel ether, chloroform and benzene, giving positive reaction to ninhydrin, Elson-Morgan and Molisch reagents but negative reaction to Sakaguchi, Tollens, Fehling, Millon and Benedict reagents, and an aqueous 1 percent solution of this substance shows a specific rotation [0111) plus 1 18". An N-acetylate obtained by acetylating 2'-amino-2'-deoxy-kanamycin with methanol-acetic anhydride has the empirical fonnula C l-l N 0, '5(Cl-l CO)'l-l 0, melts at 290-300 C. with decomposition and gives a specific rotation of [awplus 1 18.

The antibacterial activity of 2-amino-2-deoxy-kanamycin is heat stable. When a solution of the 2'-amino-2'-deoxy-kanamycin in oN-hydrochloric acid has been degraded by heating at 100C. for 40 minutes, the antibacterial activity against Bacillus subtilis of the degraded solution retained 60-80 of the initial potency of the solution. The degraded solution is applied in the form of a spot on a sheet of 40 cm. X40 cm. of Toyo filter paper No. 50 and then developed upwards with a solvent consisting of butanol-acetic acid-water (4:2:1). This paper chromatography shows that there occur two spots which give a positive reaction to ninhydrin and that one of the spots is also positive in the reaction to ammoniac silver nitrate.

2'-Amin0-2'-deoxy-kanamycin exhibits high and useful antibacterial activity against Gram-positive, Gram-negative and acid-fast bacteria as well as against such bacteria and strains which are resistant to kanamycin A, other known antibiotics and chemotherapetuic agents. Toxicity of 2-amino-2-deoxykanamycin is very low, as its LD, is only 190 mgJkg. in mice (intravenous injection).

Antibacterial spectrum of 2'-amino-2'-deoxy-kanamycin is tabulated in table lbelow.

TABLE 1 Minimum concentration for complete inhibi- Micro-organisms tested tion in meg/ml.

Aerobacter aerogenes MM 1 102 0.15 Alcaligenes faecalis 0.031 Bacillus agri 0.078 Bacillus subtilis ATCC 663 3 0.039 Bacillus subtilis PC1219 0.078 Diplococcus penumoniae Type 31 1D 0.31 Escherichia coli IAM1253 1.25 Klebsiella penumoniae 607 0.31 Lactobacillus arabinosus ATCC 8014 1.56 Lactobncillus fermenti ATCC 9338 0.15 Staphylococcus albus PCl 1200A 0.039 Staphylococcus aureus 2091 0.31 Staphylococcus aureus Terashima 0.015 Mycobacterium phlei No.

56 0.62 Mycobacterium phlei Sekiguchi 0.31 Mycobacterium 607 0.78 Proteus vulgaris 7.8 Pseudomonas aeruginosa 1AM 1007 0.1 Pseudomonas aeruginosa Takasaki 0.78 Pseudomonas aeruginosa Masakuro 0.39 Salmonella paratyphi A 0.62 Salmonella paratyphi B 0.62 Salmonella paratyphi C 1.25 Sarcina lutea PC] 1001 0.31

In the attached drawings:

FIG. 1 shows a curve of the ultraviolet absorption spectrum of 2'-amino-2'-deoxy-kanamycin dissolved in water at a concentration of l mg./ml. of water.

F IG. 2 shows a curve of the infrared absorption spectrum of 2-amino-2-deoxy-kanamycin in Nujol.

Referring to FIGS. 1 and 2, it is noted that 2'-amino-2- deoxy-kanamycin does not exhibit absorption of ultraviolet light from 220 milimicrons to 340 milimicrons but exhibits characteristic absorption bands at the following wave numbers in cm., 3,600, 3,000, 1,650, 1,600, 1,570, 1,140, 1,110, 1,095, 1,090, 1,060, 1,035, 960, 900, 895, 880, 845, 815, 790, 780, 765, and 725.

A culture of the parent strain 04-] of Streptomyces kanamyceticus has been deposited in the A.T.C.C. in Washington, D.C., and added to its permanent collection of micro-organisms as the deposition No. 21252. Furthermore, cultures of the mutants A-4-6, 3AG, 18-8 and 19-2 of Streplomyces kanamycelicus which may be used in carrying out the process of the second aspect of the present invention also have beendeposited in the A.T.C.C. as the deposition Nos. 21260, 21259, 21261 and 21268 respectively.

As stated hereinbefore, the mutants A-4-6, 3A0, l8-8 and 19-2 of Streptomyces kanamyceticus are characterized by having little or no ability to utilize xylose, as compared to the high xylose-utilizin g ability of their parent strain 0-4-1.

The mutant A-4-6 is further characterized in that it does not grow on sucrose-Czapek-agar plate containing no deoxystreptamine whereas the parent strain 0-1 do. The mutant 3A6 is further characterized in that it forms considerably larger colonies on a Czapek-agar plate containing 3-amino-glucose than the parent strain 04-1. The mutants 18-8 and 19-2 used according to the present invention are further characterized in that they do not or little utilize dulcitol whereas the parent strain 0-4-1 has a very much higher ability to utilize dulcitol.

These mutants were produced by using .the following method of mutation and screening. The procedures of obtaining each of the mutants A-4-6, 3A6, 18-8 and 19-2 are described below in more detail.

The mutant A 46 was produced by treating the typical strain 0-4-1 of Streptomyces kanamyceticus as follows: the aforesaid typical strain of Streptomyces kanamyceticus was inoculated to the surface of a slant of sucrose-Czapek-agar which was then incubated at 28 C. for 7 days. A platinumloop amount of the resulting culture was separated therefrom and inoculated to 10 ml. ofa liquid medium containing 1 percent corn steep liquor and 0.25 percent dried yeast at pH of 7.0 in a 70 ml. test tube. Shaken cultivation was carried out at 28 C. for 24 hours on a reciprocating shaker which reciprocated at 350 cycles per minute with amplitude of2 cm. The growing mycelia so obtained were isolated by centrifuging the culture broth for 10 minutes under gravity of 2,000 g. and discarding the supernatant liquid. The mycelium cake was washed twice with equal amounts of 0.9 percent physiological saline solution and the obtained wet mycelia were suspended in the 0.9 percent physiological saline solution to the volume of 5 ml. to which 4 ml. of a solution containing one-fourth mol. of Na HPO as added, then the mixture was added with 1 ml. of a solution of 500 mcg./ml. of the nitrogen mustard, and allowed to stand for 10 minutes. Subsequently, 1 ml. of

the resulting mixture was taken into 9 ml. of a neutralizing solution of 0.2 percent glycine and 0.17 percent Nal-lCO and the mixture was allowed to stand at room temperature for about 1 hour. The mycelium cake was isolated therefrom by centrifuge, rinsed and then the whole amount of the mycelia was transplanted into ml. of corn steep liquor medium in a 70 ml. test tube. Shaken cultivation was then performed at 28 C. for 24 hours in the same way as mentioned above. The culture was rinsed twice, diluted and then inoculated to sucrose- Czapek-agar plate which further contained 100 meg/ml. of deoxystreptamine added. Incubation was made at 28 C. for 10 days to give colonies.

Some amounts of mycelia were removed removed from each of the colonies and seeded to 10 ml. of corn steep liquor medium in a 70 ml. test tube. Shaken cultivation was again carried out at 28 C. for 24 hours in the same way as mentioned above. The resulting culture was twice rinsed, diluted and then inoculated to sucrose-Czapek-agar plate and sucrose-Czapek-agar plate which additionally contained 100 mcg./ml. of deoxystreptamine. Incubation was made at 28 C. for 7 days to give colonies. Such a strain which did not grow in the former plate but grew only in the latter plate was isolated anddesignated as the mutant A-4-6 of Streptomyces kanamyceticus. This mutant which exhibited essential demand for deoxystreptamine was found to be present at a ratio of about one colony to 100 colonies which were formed.

The mutant BAG was produced by treating the parent strain 0-4- as follows: a culture of the typical strain 0-4-1 of Streptamyces kanamyceticus was inoculated on the surface of a slant of sucrose-Czapek-agar and incubated at 28 C. for 7 days. A platinum-loop amount of the resulting culture was removed therefrom and transplanted in 10 ml. of a liquid medium containing 1 percent corn steep liquor and 0.25 percent dried yeast at pH 7.0 in a 70 ml. test tube. Shaken cultivation was made at 28 C. for 24 hours on a reciprocating shaker which reciprocated at 350 cycles per minutes with an amplitude of 2 cm. The culture was washed twice with physiological saline solution and then treated with a solution of 50 meg/ml. of the nitrogen mustard. Mycelia of the strain so treated were then inoculated to 3-amino-glucose-containing Czapek medium which contained 3 percent of 3-amino-glucose in place of 3 percent of sucrose, and shaken cultivation was then made at 28 C. for 24 hours in the same way as mentioned above. From the culture were separated by centrifuger the growing mycelia which were then inoculated to corn steep liquor medium for the purpose of improving the growth thereof. Shaken cultivation was made again at 28 C. for 24 hours in the same way as mentioned above. After the culture was washed, the resulting suspension was homogenized, diluted and then inoculated to 3-amino-glucose-Czapek-agar plate which was subsequently incubated at 28 C. for 14 days to form the colonies. As result, there were formed small-sized colonies, mediate-sized colonies and large-sized colonies. Mycelia were separated only from the large-sized colonies and then inoculated to corn steep liquor medium. Shaken cultivation was carried out again at 28 C. for 24 hours in the same way as mentioned above. After washing, the suspension was homogenized, diluted and then inoculated .to 3-amino-glucose-Czapek-agar plate, which was further incubated at 28 C. for 14 days to form colonies again. As result, such a strain which formed only large-sized colonies was isolated and designated as mutant 3AG of Streptomyces kanamyceticus. The mutants 18-8 and 19-2 were produced by treating the parent strain 0-4-1 as follows: a culture of the parent strain 0-4- of Streptomyces kanamyceticus was inoculated to the surface of a slant of starch-Czapek-agar and incubated at 28 C. for 7 days. A platinum-loop amount of the resulting culture was removed therefrom and suspended in 1 ml. of sterilized water. After the suspension was triturated, it was transplanted in a dish containing starch-Czapek-agar and then incubated at 28 C. for 10 days.

After the incubation, the dish was placed below an ultraviolet lamp (l9watt sterilization lamp as produced by Toshiba Co., Japan) and then irradiated with ultraviolet rays for 10 minutes with keeping a distance of 30 cm. between the dish and lamp. After this mutation treatment, the mycelia on the agar surface were scraped up and suspended in 10 ml. of sterilized water. The suspension was homogenized and then applied onto a starch-dulcitol-Czapek-agar (that is a Czapekagar containing 0.1 percent starch and 3 percent dulcitol) and incubated at 28 C. for 7 days. Mycelia were separated only from the small-sized colonies which slightly had grown on the agar. The mycelia were then inoculated to starch-Czapek-agar slants and incubated at 28 C. for 7 days. On these starch- Czapek-agar slants, all the colonies showed good growth. Platinum-loop amounts of mycelia were separated from each of the slants and then inoculated to dulcitol mineral agar slant (containing 1 percent dulcitol, 0.264 percent ammonium sulfate, 0.565 percent di-potassium hydrogen phosphate, 0.238 percent mono-potassium di-hydrogen phosphate, 0.1 percent magnesium sulfate hepta-hydrate, 0.00064 percent cupric sulfate penta-hydrate, 0.00079 percent manganese chloride tetra-hydrate, 0.00011 percent ferric sulfate heptahydrate, 0.00015 percent zinc sulfate hepta-hydrate and 1.5 percent agar, pH 7.0). Incubation at 28 C. for 14 days gave a predominant number of strains which showed the normal growth as well as only two strains which could not grow significantly. These two strains which could grow on the starch- Czapek-agar but could not grow on the dulcitol mineral agar have been found to exhibit the ability to produce 2-amino-2- deoxy-kanamycin at higher efficiency, and these mutants have been designated as mutants 18-8 and 19-2 of Strepromyces kanamyceticus, respectively.

The mutants Nos. 3A0, A-4-6 18-8 and 19-2 of Streptomyces kanamyceticus used according to the present invention have the following characteristics. These mutants Nos. 3AG, A-4-6, 18-8 and 19-2 are morphologically similar to each other in that the aerial mycelia make branches but not comparatively fine and usually do not produce spiral and whorl that spores are formed at the tips of aerial mycelia and are usually of an oval or cylindrical shape of generally 1.0 to 1.5 microns in width and that the surface of the spores is smooth. However, they are distinguishable from each other with respect to their conditions of the growth on culture media, their physiological actions and their ability to utilize carbon sources. The conditions of the growth, physiological actions and utilization of carbon sources of these mutants of Streptomyces kanamyceticus are summarized in tables 2, 3 and 4 below. Moreover, the differences between the typical strain 0-4- of Slreptomyces kanamyceticus and the mutants Nos. A-4-6, 3A0 18-8 and 19-2 derived therefrom 8 summarized in table 4 hereinafter, with respect to their growth conditions, physiological actions and utilization of carbon sources.

TABLE 2.CONDI'IIONS OF GROWTH ON DIFFERENT CULTURE MEDIA OF MUIANTS OF S TREP TOM YCES KANAMYCE TIC US [Observed alter 14-day incubation; incubation temperature 28 C.]

S. kanamyceticus Culture media Observations Mutant N o. A-4-6 Mutant No. 3AG Mutant N 0. 18-8 Mutant No. 19-2 Sucrose Czapek agar Growth Smooth growth, faint Smooth growth, Smooth growth, Smooth growth, dark yellow colored. deeply yellow deeply yellow yellow colored.

colored. colored. Aerial mycelium None None None None. Soluble pigmenL- Faint yellow Faint yellow Yellow Reverse side Faint greyish yellow Greyish yellow colored.

colored S. kanamyceticus Culture media Observations Mutant No. Ai6 Mutant N o. 3AG Mutant No. 18-8 Mutant No. 19-2 Glycerol agar Growth Very poor growth Smooth growth, Smooth growth, Smooth growth, orless. yellowish grey yellowish grey lightly brown colored. colored. colored. Aerial mycelium None None None Bright olive-grey colored. Soluble pigment do .do do Faint yellowish brown colored. Reverse side Faint greylsh colored. Faint greyish yellow Yellowish brown colored. colored.

Glucose asparagine agar Growth Yellowish brown to Deeply yellowish Bright deeply yellow Yellow colored.

(Krainsky). deeply yellow brown to brown colored.

colored. colored. Aerial mycelium None None Slightly faint yellow None.

colored. Soluble pigment do do None Do. Reverse side Yellowish grey Faint brown colored colored.

Glucose asparagine agar Growth Deeply yellow to Deeply yellow to Deeply yellow Dark yellow colored.

(Uschinsky). yellow colored. yellow colored. colored.

Aerial mycelium None None None N one. Soluble pigment ..do do do Do. Reverse side Yellow to faint Yellow to faint yellow colored. yellow colored.

Calcium malate agar Growth Faint yellow to White to faint White to very White to very yellow colored. yellow colored. faint yellow colored. faint yellow colored. Aerial mycelium None None None.. None. Soluble pigment Slightlydyellow do. do Do.

co re Reverse side White colored .Whitc colored Glycerol calcium malate agar Growth Yellowish brown Brown to yellowish Good growth, Good growth,

colored. brown colored. yellow colored. yellow colored.

Aerial mycelium None None None None. Soluble pigment Yellow Yellow Yellow Faint yellow. Reverse side. Faint yellow colored- Brown colored Bouillon agar Growth Colorless to greyish Colorless to greyish Colorless to grcyish Colorless to grayish 1 yellow colored. yellow colored. yellow colored. yellow colored. Aerial mycelium. None None None None. Soluble pigment. ..do do ..do. Do.

Glucose nutrient agar Growth Finely wrinkled Finely wrinkled Finely wrinkled Finely wrinkled growth, white to growth, white to growth, white to growth, white to faint yellow colored. faint yellow colored. faint yellow colored. faint yellow colored.

Aerial niyceliurn None None None.. None. Soluble pigment do do H. do Do.

Starch ammonium sulfate Growth Yellow to yellowish Yellow to yellowish Faint yellow to Faint yellow to agar. brown colored. brown colored. yellow colored. yellow colored.

Aerial mycelium None None Nonc None. Soluble pigment... .do ..do. do Do. Reverseside Faint yellow colored... Faint yellow colored.

Potato plug Growth Elevated good Wrinkled growth, Wrinkled growth, Wrinkled growth.

growth, ochcr granular surface, granular surface, granular surface, colored. faint yellowish faint yellowish faint yellowish brown colored. brown colored. brown colored. Aerial mycelium White colored. Slightly white Slightly white None.

colored. colored. Soluble pigment- None None None Do.

Carrot plug Growth Wrinkled growth, Wrinkled growth, Wrinkled growth, Poor growth.

poorer than on potato plug, granpoorcr than on potato plug, granpoorcr than on potato plug, granular surface. ular surface. ular surface. Aerial mycelium None Nonc Slightly white None.

colored. Soluble pigment do ..do None Do. Growth at 37 C No growth No growth Loefllers blood serum Growth Grey to cream Grey to cream Grey to cream Grey to cream colored growth. colored growth. colored growth. colored growth. Aerial mycellum None Nonc None None. Soluble pigment do do do Do. Growth at 37 C Similar to the 28 (1.. Similar to the 28 C. Similar to the 8 C.

growth. growth. growth. Egg Growth Wrinkled growth, Wrinkled growth, Wrinkled growth, W rinkled growth,

yellow colored. yellow colored. yellow colored. yellow colored. Aerial mycelium None N one. None None. Soluble pigment do do do Do.

Skimmed milk Growth No growth No rowth Ring growth Ring growth.

Aerial mycelium Nollie None.

Soluble pigment [Observed after the incubation for 14 days at 28 0.]

TABLE 3.PHYSIOLOGICAL ACTION OF THE MUTANT OF STREPTOMYCES KANAMYCETICUS S. kanamyceticus Physiological actions Mutant N o. A-4-6 Mutant No. BAG Mutant No. 18-8 Mutant N 0. 19-2 Reduction of nitrate Positive Positive Positive. Positive. Hydrolysis of starch d ...do Do. Formation-of hydrogen sulfide- N egativc. Negative Formation of tyrosinase do v Do. Liquefaction of gelatin (at 15 C. for 14 days) Strongly liquefied Liquefied... Liquefied. olubilisation of Loefliers blood serum (at No observed. No observed No observed.

28 C. and 37 C. for 14 days) Skimmed milk medium N o coagulation, slow peptonization.

No coagulation, slow pcptonization.

TABLE 4.DIFFERENCES BETWEEN THE TYPICAL STRAIN -4-1AND THE MUIANTS 0F STREPTOMYCES KANAMYCEQ I 0 US S. kanamyceticus Mutant Mutant Mutant Mutant Observations Strain 041 No. A-4-6 No. BAG No. 18-8 No. 19-2 Growth on:

Sucrose Czapek agar plate 1 Deoxystreptamine Czapek agar plate. :1: :l: =l=

B-aminoglueose Czapek agar plate i Dulcitol Czapek agar plate d: i

P tato plug a a 3 Physiological action:

Liquefaction of gelatin In skimmed milk medium (t 4 (4) Utilization of carbon sources:

Su r se :1:

Xylose d: 3:

Sodium acetate d:

Sodium citrate Sodium succinate" 5:

Dul it l i i 3 wrinkled growth, granular surface, faint yellowish brown colored.

4 Slow geptonization. 5 Rapi peptonization.

EXAMPLE 1 A liquid culture medium 1.) containing 3.0 percent maltose, 2.0 percent starch, 3.0 percent soybean meal, 1.0 percent sodium nitrate and 0.5 percent sodium chloride was inoculated with the mutant No. A-4-6 of Streptomyces kanamyceticus and then incubated at 28 C. under aeration and agitation. The quantity of 2'-amino-2'deoxy-kanamycin accumulating the culture reached maximum at the end of 6 days of the incubation.

The culture broth was then filtered to give 14 l. of the filtrate which was then treated with 4 l. of Amberlite lRC-SO (ammonium type). The resin was subsequently eluted with 0.5 N aqueous ammonia. The active fractions were collected and concentrated to a syrup which was then dissolved in 500 ml. of water. The solution was decolorized by treating with 6 g. of active carbon. The solution was filtered to remove the carbon, and the filtrate was subjected to chromatographic elution with 600 ml. of Dowex 1X2 (OH type). 2'-Amino-2-deoxy-kanamycin was eluted out before the separation of kanamycin A. The fractions containing high concentration of 2'-amino-2'- deoxy-kanamycin were collected, concentrated and freezedried to give 3.2 g. of a crude powder. This crude powder was dissolved in 50 ml. of water and the solution was treated with a resin Amberlite CG-SO (ammonium type) to adsorb the antibiotics. The resin was again subjected to chromatographic elution with 0.1 N to 0.5 N aqueous ammonia. The coexisting small amount of kanamycin A was then eluted out at first and 2'-amino-2'-deoxy-kanamycin was subsequently eluted separately as a single fraction. This single fraction was concentrated and freeze-dried to give a crude powder of 2-amino-2'- deoxy-kanamycin. Recrystallization of this powder from water-dimethyl-formamide gave 1.9 g. of 2'-amino-2-deoxykanamycin in the form of colorless, needlelike crystals.

EXAMPLE 2 A liquid culture medium (15 1.) having the same composition as in example 1 was inoculated with the mutant No. BAG of Streptomyces kanamyceticus and then incubated at 28 C. under aeration and agitation. The quantity of 2'-amino-2'- deoxy-kanamycin accumulating in the culture reached maximum at the end of 6 days of incubation.

The culture liquid was then treated in the same procedures as in example 1 to give 1.2 g. of a crystalline form of a2- amino-2'-deoxy-kanamycin.

EXAMPLE 3 A liquid culture medium (15 1.)having the same composition as in example 1 was inoculated with the mutant No. 18-8 of Streptomyces kanamyceticus and then incubated at 28 C. under aeration and agitation. The quantity of 2'-amino-2- deoxy-kanamycin accumulating the culture reached maximum at the end of 7 days of incubation.

The culture liquid was then treated in the same procedures as in example 1 to give about 2.2 g. of a crystalline form of 2'- amino-2-deoxy-kanamycin.

EXAMPLE 4 The procedure of example 1 was repeated using the mutant 30 No. l9-2 of Streplomyces kanamyceticus. A similar result was kanamycin was obtained in a crystalline form.

EXAMPLE 5 (comparative) In this example, a series of tests was carried out in order to demonstrate the unexpectable and advantageous effect of using the mutants A-4-6, 3A6, 18-8 and 19-2 which intrinsically have the higher ability to produce 2-amino-2'-deoxykanamycin, on the production of 2'-a mino-2'-deoxy-kanamycm according to the present invention, as compared to when the parent strain 0-4-1 of Streptomyces kanamyceticus is cultivated in a culture medium.

In these tests, the cultivation of the parent strain o-4-l and the mutants A46, 3A0, 18-8 and 19-2 of Streptomyces kanamyceticus was carried out at 28 C. for 150 hours in the same manner as in example 1 using similar jar fermentors and the culture media of the same composition, respectively. After the end of the cultivation, the resulting culture broths were assayed for the content of 2-amino-2'-deoxy-kanamycin and of kanamycin A. The results of assay are shown in table 5 below.

Each 15 litres of the resulting culture broths were then treated in the same manner as in example 1 so that 2'-amino-2 -deoxy-kanamycin and kanamycin A could be recovered therefrom as much as possible. Amounts of the products recovered are shown in table 6 below.

obtained to that of example 1. 3.15 g. of 2'-amino-2'-deoxytants A4-6, BAG, 18-8 and 19-2 have the intrinsic ability to produce 2'-amino-2'-deoxy-kanamycin in a remarkably higher yield than the parent, typical strain 0-4-1 of Streptomyces kanamyceticus.

What we claim is:

1. A process for the production of 2-amino-2'deoxy-kanamycin in higher yield, which comprises cultivating the mutant selected from the group consisting of mutants identified as ATCC 21259, ATCC 21260, ATCC 21261 and ATCC 21268 of Streptomyces kanamyceticus under aerobic conditions in a usual culture medium containing carbohydrate and nitrogenous nutrient until 2'-amino-2-deoxy-kanamycin accumulates in a profitably large amount in the culture, and then recovering 2 amino-2-deoxy-kanamycin from the culture. 

